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Salmonellae are of major importance as causes of food borne disease. Classical diagnostic methods that rely on cultivation, Widal slide agglutination tests and biochemical tests for detecting Salmonella are time-consuming. More rapid identification is highly desirable for clinical and epidemiological reasons. This obstacle can be overcome by Fluorescence in situ Hybridization (FISH). FISH uses fluorescently labelled probes that hybridize specifically to complementary target sequences on the bacterial ribosomal RNA within the bacterial cell. In this study six newly developed probes specific for Salmonella enterica and three already publicized probes were tested and evaluated on reference strains and more than 200 clinical isolates. Two of the new and one of the publicized probes were very sensitive and specific. The two new probes showed cross reactions to different bacteria strains. If only concordant positive results were regarded as true positives the FISH method was 100 % specific. Moreover, a procedure was established for the detection of Salmonella directly in stool samples by FISH. The analytical sensitivity of FISH in comparison with conventional culture was assessed on stool samples spiked with serial dilutions of Salmonella. FISH worked for the detection of Salmonella directly in stool samples, but culture was more than 100 times more sensitive. Finally the oligonucleotide probes were used for the direct identification of Salmonella spp. in 207 positive blood cultures. Except one Serratia sp. no false-positive results have occurred and each Salmonella spp. was correctly identified. In conclusion FISH is a useful method for fast, cheap and easy identification of bacterial isolates as Salmonella enterica. FISH can confirm microbiological culture but it will not replace culture-based methods in the near future. For identification of Salmonella spp. in positive blood cultures FISH proved to be a very sensitive and specific method.